Association of Mannose-Binding Lectin 2 (MBL2) gene heterogeneity and its serum concentration with osteoporosis in postmenopausal women

Th e aim of the study was to detect prevalence of MBL exon  (codons ,  and ) genetic polymorphism in postmenopausal women in Bosnia and Herzegovina and its possible role as genetic risk factor for susceptibility to occurrence of osteoporosis in this study group. Also, we investigated association between MBL serum concentrations and osteoporosis in postmenopausal women. Genetic codons’ variations were determined by PCR-RFLP and MBL in serum was measured by ELISA method in  postmenopausal women ( with osteoporosis and  apparently healthy, non-osteoporotic women serving as a control). Serum MBL levels were not signifi cantly diff erent between osteoporosis and control group ( (-.) and . (-.) ng/mL respectively, p=.). Genotype frequencies were not signifi cantly diff erent (p=.) between the studied groups of postmenopausal women. Genotype frequencies A/A, A/ and / in osteoporosis group were .; .; . and in control group .; .; ., respectively. Frequencies of A and  allele were . and . in osteoporosis and . and . in control group. Th e results do not suggest association of functional polymorphism of MBL gene and MBL serum concentration with osteoporosis in postmenopausal females. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Mannose-binding lectin (MBL) is in focus of attention because of its role as a recognition molecule in complement system.It is Ca + -dependent collagenous lectin, synthesized in liver with main role to mediate innate immune defence against microorganisms.MBL recognizes certain sugars on the surface of the bacteria, apoptotic cells, phospholipids and immune complexes and mediates opsonophagocytosis directly and by activation of lectin complement pathway [].MBL is also considered as acute-phase reactant and its responsiveness is dependent upon the MBL genotype [].
Serum MBL concentration varies from undetectable to . ng/mL.It is very well documented that decreased concentration of MBL is associated with susceptibility to infectious diseases.Furthermore, lower concentration may be caused by point mutation on exon  and by polymorphism on promoter region of MBL gene [].It is clear that MBL gene harbor complex genetic system associated with infectious conditions but also it may be a diseases modifier in patients with certain diseases.Certain aspects of osteoporosis have genetic infl uence [].Since, the osteoporosis shows an inflammatory character [] and MBL is considered as modifi er of infl ammatory responses, we investigated association of MBL gene heterogeneity and MBL serum concentration with osteoporosis.It was shown that serum level of MBL is infl uenced by presence of genetic polymorphism at the protein coding region consist of four exons [].MBL gene is located in long arm of the chromosome q.-q[].MBL is pseudo gene.Five functional single-nucleotide polymorphisms can be found in the MBL gene.Each of them can affect se-rum levels of the MBL.Two polymorphisms are located within the promoter region of the gene (at - and at -) and  other at first exon of coding region.These functional polymorphisms of the MBL gene result in single amino acid substitutions that reduce functional levels by causing structural defects in the MBL protein.
Th ree independent point mutations had been reported: substitution of arginine with cysteine at codon  (allele D), glycine with aspartic acid at codon  (allele B) and glycine with glutamic acid at codon  (allele C) [].Th e common designation for these three mutation variant is 0 (zero) and wild type allele has been named as A. Th ese three mutations disrupt the assembly of the MBL oligomeric molecules; codon variants  and  disrupt the Gly-X-Y repeats in the collagenlike domain and codon  disrupts the N-terminal disulphide bonds between primary  kDa MBL structural units.So, these variants prevent forming tertiary structure of MBL [].MBL functional polymorphism has been studied in wide variety of pathological conditions (from infectious to autoimmune).Still role of MBL in susceptibility to diff erent diseases is controversial.According to many authors, for the same diseases, fi ndings of association or lack of association mainly depends on ethnicity of subjects included in studies.

Study population
This cross-sectional study included  postmenopausal women ( with osteoporosis (.±.years) and  apparently healthy, non-osteoporotic women serving as a control (.±.years).All women underwent bone mineral density (BMD) assessment at the hip and lumbar spine which was performed by Dual-energy x-ray absorptiometry (DXA) at Clinics for Radiology, Clinical Center University of Sarajevo.Osteoporosis was defi ned as total T score equal or below -. measured either on the hip and/or lumbar spine.Women whose total T score was - or higher, both on the hip and/or lumbar spine were considered to have preserved bone mass and served as controls.The Ethical Committee of Faculty of Medicine, University of Sarajevo approved the protocol of the study.Sample collection and all laboratory procedures were done in the Laboratory for Molecular medicine, Center for Genetic, Faculty of Medicine, University of Sarajevo.Written informed consent was obtained from each subject included in this study.To obtain genomic DNA for genetic testing, buccal cells were collected with two swabs.Subject's mouth was vigorously rubbed on the both sides of the cheek at least six times and swabs were placed inside of envelope.Used cotton swabs and the envelope were sterile.Upon receipt, the buccal swabs were placed at room temperature to dry, and keep at - o C until DNA extraction.Genomic DNA was isolated from buccal swabs following standard salting out procedure (Miller) [].

Genotyping of MBL
Detection of the genetic polymorphism in codons ,  and  of the MBL gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the restriction enzymes HhaI, BanI and MboII, respectively.For determination of polymorphisms following pair of primers were used: MBLexF '-CAT CAA CGG CTT CCC AGG CAA AGA TGC G-' and MBLexR '-CAG GCA GTT TCC TCT GGA AGG TAA AG-' as reported previously [].The PCRs were performed in final volume of  μl using  ng of genomic DNA, . pM of forward and reverse primer each (Eurofins MWG Operon, Germany) . mM of MgCl,  U of Taq polymerase in buffer containing  mM Tris-HCl (pH .) and  mM KCl (Qiagen, Germany) and  μM of dNTP (Sigma-Aldrich Chemie GmbH, Germany).PCR conditions were as follow: an initial denaturation at °C for  min,  cycles of denaturation at °C for  s, annealing at °C for  s, and extension at °C for  s.Th e PCR was followed by a final step at °C for  minutes.Obtained PCR product with this procedure was  pb.Amplifi ed products were cleaved with restriction enzyme (TAKARA BIO INK, Japan) HhaI into  and  bp for the A allele and were uncleaved when the D variant is present.Fragments with wildtype, for codon , were cleaved into two fragment of  and  bp, but with B allele stayed undigested by BanI.For codon , fragments with A variant stayed uncut by MboII, while fragment with mutant allele showed two bands of  and  bp.Th e genotypes were determinate by electrophoresis on . agarose gel stained with ethidium bromide.

Measurement of MBL
Venous blood samples were taken and centrifuged at  g for  minutes.Collected serum samples were stored at - o C. Quantitative determination of serum MBL levels was performed by a solid-phase enzyme-linked immunoassay (ELI-SA).Samples were diluted and processed according to the manufacturer's instructions (HyCult Biotechnology).Briefl y, after activation of samples and standards, they are incubated in microtiter wells coated with mannan.After capturing of human MBL, biotinylated tracer antibody, streptavidin conjugate and tetramethylbenzidine (TMB) in separate phases of protocol were added.Th e reaction was stopped by oxalic acid addition.Sample MBL levels were calculated from the standard curve based on samples of known MBL concentration.Absorbances were read at A by using ELISA reader (STAT FAX , USA).Values of MBL were expressed in ng/ml.

RESULTS
Seventy five participants included in the study were divided into two groups; women with osteoporosis and healthy control.Initially, evidence of the presence of the MBL allele variants B, C and D was sought in DNA samples from postmenopausal women (Figure .).Table  presents the genotype frequencies of the exon  variants of MBL in postmenopausal women with osteoporosis and control group.There were no statistically significant differences regarding genotype frequency of the MBL gene polymorphism (p=.) in studied groups.The genotype frequencies of A/A, A/ and / were .; .; . in osteoporosis and .; .; . in control group of postmenopausal females.Th e data of allele frequencies of point mutations on MBL gene in postmenopausal women are presented in Table .Th e frequency of A variant allele was ., but  allele variant frequency was ..Allele variant frequencies for three diff erent structural variants were ., . and . for B, D and C respectively.Concerning the osteoporosis and control groups, frequencies of the A allele were . and . and of  allele were . and ., respectively.MBL serum concentrations in postmenopausal women were shown in Table .Comparison of mannose-binding lectin serum concentration between postmenopausal women with osteoporosis and control shown no signifi cant diff erence ( (-.)ng/mL and . (-.)ng/mL respectively, p=.).Analysis of the MBL serum concentration and corresponding MBL gene polymorphism in postmenopausal women confi rmed that genotype variants have eff ects on the concentration of MBL.Mannose-binding lectin concentration in serum of postmenopausal women was lower in variant alleles (.(-.)ng/mL) compared to wild-type allele carriers (.(.-.)ng/mL) (p=.).After distribution of participants into two groups (women with osteoporosis and group with preserved bone mass -control), it was shown that the wild-type MBL genotype AA was associated with high MBL level (.(-.)ng/ mL) while, A/ genotype was associated with lower concentration of MBL in serum (.(. -.)ng/mL) in a group of women with osteoporosis (p=.).Th is genotype infl uence was not confi rmed in control group (p=.).Obtained frequencies of the allele B, C and D variants were higher in comparison with results of Garred et al. [] who reported lower frequencies in the healthy European Caucasians (., . and . respectively).Also, allele frequencies were higher compared with the results from United States population-based epidemic study which enrolled Non-Hispanic Whites (., . and . respectively) [].The cross-sectional study carried out in West African region conducted by Mombo et al. [] presented allelic frequencies of MBL variant B and variant C (. and .).Frequencies of B and C allele variants of subjects included in this study were similar to the latter results.Th e different distribution of genotypic and allelic frequencies in diff erent population is a result of ethnic diff erence [].
These study results of allele and genotype frequencies of MBL gene variants were in compliance with results of Yarden et al. [] who included European Caucasian patients with cystic fi brosis and control subjects (., . and . for AA, A and  for genotype and . and . for allele frequencies respectively).Also, genotype frequencies for all postmenopausal women in this study (Table .) were similar to the frequencies found in a control group in the study by Nielsen et al.
[] (. for AA, . for A and . for  genotype groups).Th ere is no evidence regarding gender diff erence in the genetic variation in MBL gene currently available in the literature in healthy people or in people with certain diseases [,], so this study results of genotype and allele frequencies in female population cannot be compared with published data.
It is clear that MBL gene harbours complex genetic system associated with infection conditions but also it may be considered as a diseases modifi er in patients with certain diseases [].Obtained results showed that the polymorphisms of MBL gene did not seem to be associated with osteoporosis.
No diff erences were found in number of subject with heterozygous or homozygous mutation of all three codons in postmenopausal women.Increased frequency of MBL genotype in women with osteoporosis comparing to control was not found.Th e frequencies of exon  variation in this study were obviously similar to control group of women.Since study included a small number of women, further studies with larger sample size should be performed to clarify possible association between osteoporosis and MBL gene polymorphism.
As it is already reported, MBL polymorphism in exon  causes lower MBL serum concentration [, ].Th ese study results confi rmed the infl uence of diff erent MBL genotypes on MBL concentration in postmenopausal women.Inconsistent result was observed when we tested this association between two study groups.Infl uence of polymorphism on MBL concentration in serum was observed only in osteoporosis group.Signifi cantly higher MBL concentration was detected in women of osteoporosis group carrying wild-type genotype comparing to allele variant carriers.The influence of genotype on serum MBL levels in a healthy group of women was not confi rmed, since no diff erence in MBL concentration was found among diff erent genotype carriers.
There is strong correlation of MBL polymorphism and MBL concentration and function, confi rmed by many studies [, ].Despite these obvious associations, substantial interpersonal variations of the MBL concentration have been observed.Many individuals with wild-type MBL genes may have low or undetectable MBL levels and function [].Also, variations of MBL concentration between the same genotype have been observed [].Diff erence in circulating serum concentration of MBL cannot be explained just by infl uence of MBL gene polymorphism in exon  but there is also signifi cant infl uence of the polymorphism in promoter region [].Investigation of promoter region genetic variation haven't been included in this study, so large research has to be done to elucidate mechanism and association of MBL  gene polymorphism and circulated serum MBL level.
In conclusion, we could not find an association of MBL polymorphism with susceptibility to occurrence of osteoporosis in postmenopausal women.Also, lack of the Bosn J Basic Med Sci 2014; 14 (1): 26-29Statistical analysisGenotype and allele frequencies were obtained by direct counting.Genotype frequencies diff erences were analyzed by Pearson χ  test.In descriptive statistic median with quartiles (fi rst and third) were used.Non-parametric Mann-Whitney U-test was used to compare continuous data between carriers of the diff erent MBL genotypes.Statistical signifi cance was defi ned as p<..Statistical calculation was performed with SPSS for Windows (version ..SPSS Chicago, IL).

TABLE 2 .
Allele frequencies of three diff erent point mutation on structural MBL2 gene in postmenopausal womenA -wild-type MBL2 allele; B-the codon 54 allele; C-the codon 52 allele; D -the codon 57 allele; 0 -any combination of the structural variant alleles;

TABLE 1 .
Genotype In presented study of postmenopausal women, examined frequencies of the genotype with structural variant alleles of MBL gene (A/) were ..Th is result is in accordance with results of Garred et al.[]who shown similar genotype frequency for Danish and British Caucasian population (.).The three allele variants of MBL gene show up with different frequencies in different population.The B allele frequency (.) in this study group was higher comparing to the C allele (.).A frequency of D allele (.) was more frequent then the C allele variant.

TABLE 3 .
Serum MBL concentrations in postmenopausal women with osteoporosis and control groupA -wild-type MBL2 allele; 0 -any combination of the structural variant alleles B, C or D; A0/00 -heterozygous and homozygous exon 1 variants; p -probability.Data are presented as median level and with fi rst and third quartile.NS -non-signifi cant diff erence osteoporosis vs. control regarding the association between MBL serum concentration and osteoporosis was observed.Genetic influence of structural MBL gene on MBL concentration was confirmed in group of postmenopausal women with osteoporosis.Study with larger number of participants and further assessment of polymorphism in the promoter region of the MBL gene is necessary to distinguish whether there is association between MBL gene polymorphisms, serum MBL concentration and osteoporosis.